Biodiversity Data Journal 9: e68016 doi: 10.3897/BDJ.9.e68016 Taxonomic Paper A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) in Japan Hiroki Matsuda‡,§, Ayman Khamis Elsayed|, Wanggyu Kim ¶, Satoshi Yamauchi#, Martin Libra ¤,«, Naoto Kamata»,˄ ‡,Junichi˅ Yukawa , Makoto Tokuda ‡ Laboratory of Systems Ecology, Faculty of Agriculture, Saga University, Saga, Japan § IDEA consultants, inc., Yokohama, Japan | The Botanical Gardens, Graduate School of Science, The University of Tokyo, Tokyo, Japan ¶ Animal Resources Division, National Institute of Biological Resources, Incheon, Republic of Korea # Shinjo, Aomori, Japan ¤ Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic « Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Ceske Budejovice, Czech Republic » The University of Tokyo Hokkaido Forest, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Furano, Japan ˄ Entomological Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan ˅ The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan Corresponding author: Makoto Tokuda ([email protected]) Academic editor: AJ Fleming Received: 28 Apr 2021 | Accepted: 28 May 2021 | Published: 17 Jun 2021 Citation: Matsuda H, Elsayed AK, Kim W, Yamauchi S, Libra M, Kamata N, Yukawa J, Tokuda M (2021) A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) in Japan. Biodiversity Data Journal 9: e68016. https://doi.org/10.3897/BDJ.9.e68016 ZooBank: urn:lsid:zoobank.org:pub:64D5D12E-DBCE-429F-B4E1-D398EB1C60AA Abstract Background A gall midge species (Diptera: Cecidomyiidae) inducing leaf bud galls on Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) was found in Hokkaido and northern Honshu, Japan. © Matsuda H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Matsuda H et al New information Based on its morphology, the species is regarded as an undescribed species of the genus Pseudasphondylia Monzen (Cecidomyiinae, Cecidomyiidi, Asphondyliini). The species is herein described as Pseudasphondylia saohimea Matsuda, Elsayed and Tokuda sp. n. The new species is easily distinguishable from its congeners by the number of adult palpal segments and the shape of the male terminalia and larval spatula. Keywords Asphondyliini, Asphondyliina, gall midge, molecular phylogeny Introduction The genus Pseudasphondylia (Diptera: Cecidomyiidae: Asphondyliini) includes 11 species associated with various plant families (Gagné and Jaschhof 2021). Nine of them are distributed in the eastern Palearctic Region (e.g. Elsayed et al. 2019, Tokuda and Yukawa 2005), one in India and one in New Caledonia (Gagné and Jaschhof 2021). Amongst the nine eastern Palaearctic species, the following six species have been recorded from Japan: P. elaeocarpi Tokuda and Yukawa on Elaeocarpus zollingeri K.Koch (= E. sylvestris var. ellipticus Hara) (Elaeocarpaceae), P. kiritanii Tokuda & Yukawa on Cornus controversa Hemsl. ex Prain (Cornaceae), P. matatabi (Yuasa & Kumazawa) on Actinidia polygama (Siebold et Zucc.) Planch. ex Maxim. (Actinidiaceae), P. neolitseae Yukawa on Neolitsea sericea (Blume) Koidz. (Lauraceae), P. rokuharensis Monzen on Viburnum dilatatum Thunb. (Caprifoliaceae) and P. tominagai Elsayed & Tokuda on Eleutherococcus spinosus (L.f.) S.Y.Hu (Araliaceae) (Monzen 1955, Tokuda 2012, Tokuda and Yukawa 2002, Tokuda et al. 2007, Yukawa 1974, Yukawa and Masuda 1996, Elsayed et al. 2019, Yukawa 1983). They have type IIA or IIB life history strategy, in which mature (type IIA) or immature (type IIB) larvae overwinter in galls on their host plants (Yukawa 1974, Yukawa 1987, Tokuda and Yukawa 2005, Tokuda et al. 2007). In addition, some species exhibit polymodal emergence patterns caused by long-term diapause (Takasu and Yukawa 1984) and other species are suspected to alternate host plants (Yukawa and Masuda 1996, Tokuda and Yukawa 2005, Elsayed et al. 2019a). In recent years, we found an undescribed species of asphondyliine gall midge that induces leaf bud galls on Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) in Tomakomai, Hokkaido and Aomori Prefecture, Honshu, Japan. Based on morphological comparison, we concluded that the gall midge is a member of Pseudasphondylia. We describe the species as new to science and discuss the phylogenetic relationships amongst Japanese congeners on the basis of molecular analysis. We also compare its life history strategy with those of the Japanese congeners. A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with ... 3 Materials and methods Collecting and Rearing Leaf-bud galls on M. kobus var. borealis (Fig. 1) were collected from Tomakomai City, Hokkaido and Mutsu City, Aomori Prefecture, Honshu, Japan. Some galls were dissected under a stereoscopic microscope to obtain mature larvae and others were kept in plastic bags until emergence of adults. Most specimens were preserved either in 75% ethanol for morphological examinations or in 99.5% ethanol for molecular analyses. Figure 1. Leaf galls induced by Pseudasphondylia sahohimea sp. n. on Magnolia kobus var. borealis. Morphological Examination and Terminology Gall midge specimens were mounted on slides in Canada balsam, following the technique outlined in Gagné (1994), except for the clearing step for the larval and adult specimens following Elsayed et al. (2018). The slide-mounted specimens were examined under a bright-field and phase-contrast microscope (H550L, Nikon, Tokyo) and line illustrations were made following Elsayed et al. (2020) under a bright-field and phase-contrast microscope (CX43, Olympus, Tokyo). Photomicrographs were taken with a digital camera (DP22, Olympus, Tokyo) attached to a semi-motorised fluorescence microscope (BX53, Olympus, Tokyo). Morphological terminology basically follows Gagné (2018), except the term “antennal papillae” in pupa follows Elsayed et al. (2020a). Larval and pupal terminology follow Gagné (1994). The holotype and paratypes are deposited in the collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (ELKU). A generic synopsis of Pseudasphondylia is presented in Tokuda and Yukawa (2005). 4 Matsuda H et al Molecular Phylogenetic Analysis Genome DNA was extracted from 99.5% ethanol-preserved female gall midges reared from leaf galls of M. kobus var. borealis and of P. tominagai reared from flower bud galls of E. spinosus (Elsayed et al. 2019) and from adults of P. elaeocarpi obtained from leaf galls on E. sylvestris var. ellipticus (Elaeocarpaceae). A 472 bp fragment of the mitochondrial gene cytochrome oxidase subunit I (COI) of the species from leaf galls of M. kobus var. borealis and P. tominagai was sequenced and aligned following Elsayed et al. (2017), using the following primer set: Forward: J-1718 (5’–GGA GGA TTT GGA AAT TGA TTA GTT CC–3’) (Simon et al. 1994) and reverse: COIA (5’–CCC GGT AAA ATT AAA ATA TAA ACT TC–3’) (Funk et al. 1995), while a 439 bp fragment of the same gene of P. elaeocarpi was sequenced using the primer set: COIS (5’–GGA TCA CCT GAT ATA GCA TTC CCA TAT TGG–3’) and COIA (5'–CCC GGT AAA ATT AAA ATA TAA ACT TC–3’) (Funk et al. 1995). The sequences obtained were compared using MEGA (version X) (Kumar et al. 2018) and deposited in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL) and GenBank nucleotide sequence databases as accession numbers LC621302–621303, LC621304–621305 and LC621306 for P. tominagai, the gall midge of M. kobus var. borealis and P. elaeocarpi, respectively. Besides the obtained sequence data, sequences of four species of Pseudasphondylia and four species of Asphondylia were downloaded from the GenBank and used as ingroup taxa: P. rokuharensis (LC538357), P. kiritanii (LC538356), P. matatabi (AB085873) (Lin et al. 2020), P. neolitseae (AB334237) (Tokuda et al. 2008), Asphondylia tojoi Elsayed and Tokuda (LC373200) (Elsayed et al. 2018b), A. aucubae Yukawa & Ohsaki (AB238595) (Uechi and Yukawa 2006b), A. yushimai Yukawa and Uechi (AB194473) (Uechi et al. 2005) and A. sphaera Monzen (AB197945) (Uechi and Yukawa 2006a). GenBank sequences of two distant species were used as outgroup taxa: Ampelomyia conicocoricis Elsayed and Tokuda (LC422091) (Elsayed et al. 2019b) and Gephyraulus zewaili Elsayed and Tokuda (LC270942) (Elsayed et al. 2017a). These sequences were aligned using ClustalW algorithm in MEGA X. We inferred the phylogenetic relationships in the same software using the Maximum Likelihood (ML) method and 1000 bootstrap replications, based on the GTR+I model determined by jModelTest 2 (Darriba et al. 2012, Guindon and Gascuel 2003). Taxon treatment Pseudasphondylia saohimea Matsuda, Elsayed & Tokuda, sp. n. • ZooBank C7679EFB-7E8C-4157-A5AE-C618D2D82B28 Materials Holotype: a. order: Diptera; family: Cecidomyiidae; taxonRank: species; nomenclaturalCode: ICZN; genus: Pseudasphondylia; specificEpithet: saohimea; country: Japan; stateProvince: Aomori; locality: Ashizaki, Mutsu City; samplingProtocol: reared from a leaf bud gall on A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with ... 5 Magnolia kobus var. borealis (Magnoliaceae) collected on 10.v.2015